Method of preparing activated killer monocytes for treating colorectal cancer

ABSTRACT

The present invention discloses a method of preparing activated killer monocytes for treating colorectal cancer. Activated killer monocytes are prepared in serum free medium in polypropylene containers.

This is a continuation in part of the application Ser. No. 06/743,570filed June 11, 1985, now abandoned.

TECHNICAL FIELD

The present invention is related generally to cancer therapy. Moreparticularly, the present invention is related to monitoring in cancerpatients the tumoricidal activity of purified human monocytes culturedin suspension in a serum-free medium.

BACKGROUND OF THE INVENTION

Mononuclear phagocytes (monocytes) in their various forms have beenshown to participate in many critical phases of the mammalian immuneresponse. Monocytes and macrophages are known to be essential for theinitiation of immune responses by virtue of their ability to processantigen (Rosenthal, New Engl. J. Med. 303, 1153. 1980), and for theirability to secrete soluble factors such as interleukin 1 (IL-1), colonystimulating factor (CSF), interferon (IFN) and prostaglandin E (PGE)which allow them to function as immunoregulators for a number of immuneresponses (Epstein, Biology of Lymphokines; Academic Press, NY, pp.123-152. 1979; Stevenson, The Reticuloendothelial System. AComprehensive Treatise, Vol. VI: Plenum Press, NY, pp. 79-91. 19882). Inaddition, monocytes are known to play critical role as final effectorcells in humoral immunity by virtue of the fact that these cells secretecomplement components (Nathan, et al, New England J. Med. 303, 623.1980) and are capable of mediating cytotoxic functions. In addition toantibody-dependent cellular cytotoxicity (ADCC) (Poplack, et al, Blood48, 890. 1976), activated killer monocytes (AKM) are known to be potentkillers of tumor cells (Stevenson, et al, Artificial Organs 112, 128.1988).

Assessment of the in vitro function of human monocytes and AKM has beenhampered by a number of technical and theoretical problems. First,monocytes constitute a very low proportion of the cells in humanperipheral blood (generally less than 5%); thus, obtaining large numbersof them has been quite difficult. In addition, very few techniques haveemerged which allow for the large-scale isolation of purifiedpopulations of human monocytes by negative selection; instead, generallysmall numbers of rather impure monocytes are isolated on gradients suchas Percoll (Hester, et al., 1981) or cells of higher purity are obtainedby adhering them onto plastic or glass labware by positive selection(Werb, J. Exp. Med. 147, 1695. 1978).

When monocytes are obtained by positive selection, it is difficult toremove them for further study; a variety of rather harsh measures areutilized to remove the adhered cells from the plastic or glass surface,ranging from the use of rubber policemen (Pennline. Manual of MacrophageMethodology, pp. 65-77. 1981), EDTA (Ackerman and Douglas, J. Immunol.120, 1372. 1979), and lidocaine-containing solutions (Koski, et al, InVitro Methods in Cell-Mediated and Tumor Immunity, Academic Press, p359. 1976). The potential problems of adherence and positive selectionare compounded when the cells are activated in culture into AKM sincemost researchers employ some sort of polystyrene labware to which themonocytes will readily adhere.

Adherent monocytes, of course, are in a different condition from theirnormal state of suspension in human peripheral blood. Therefore, thefunctions may also be different. Furthermore, when placed in mediumconditions under which they are generally cultured, human monocytesdemonstrate a number of technical inadequacies. These problems mainlystem from the limited nutrients provided in most standard laboratoryculture media for a cell as metabolically active as the human monocyte,and the additional potential artifacts created by culturing humanmonocytes with sera from different human individuals (AB serum) or fromother species (such as fetal calf serum). Thus consistent and uniformconditions for culturing human monocytes and transforming them into AKMcannot be assured from batch to batch.

The above-cited problems regarding the handling of human monocytes arecompounded when these cells are transformed into activated killermonocytes (AKM) for the treatment of cancer. Prior to the presentinvention, it has not been possible to prepare AKM in suspensionsuitable for administration to cancer patients for therapy. Moreover, nomethod for monitoring the cytotoxic activity of serum-free, suspensioncultured AKM in vitro existed.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to providesubstantially pure, clinical grade, functional, human, AKM produced insuspension in polypropylene were in a serum free medium.

It is a further object of the present invention to provide an assay formonitoring the tumorical function of isolated, substantially pure, humanmonocytes cultured in suspension in serum-free medium and transformedinto activated killer monocytes (AKM).

It is an additional object of the present invention to provide a methodof treating cancer in mammals comprising administering to said mammal animmunotherapeutic amount of isolated, purified monocytes suspended in aserum-free medium and transformed into AKM with suitable adjuvantsand/or biological response modifiers.

Other objects and advantages of the present invention would becomeapparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

FIG. 1 displays the enhanced tumor cell killing capability of AKMcultured under serum-free and suspension culture conditions.

FIG. 2 is a scanning electron micrograph showing superior killinginteractions between AKM ( ) and tumor target cells (↑) in vitro underpolypropylene (C&D) versus polystyrene (E&F) culture conditions.

FIG. 3 shows enhanced effectiveness of AKM killing of a variety of humantumor targets under polypropylene versus polystyrene culture conditions.

FIG. 4 shows the first representation of tumor cell killing by AKMdepicted by the lytre unit method, an achievement possible only underserum-free suspension culture conditions of the present invention.

FIG. 5 shows a flow sheet of procedures involved in the AKM protocol forperitoneal colorectal carcinomatosis; and

FIG. 6 (A-B) and 7 (A-D) show the distribution of ¹¹¹ Indium-labeled AKMin the peritoneum 4 hours after infusion into a peritoneal colorectalcarcinomatosis patient.

DETAILED DESCRIPTION OF THE INVENTION

These and other objects and advantages of the present invention areachieved by providing isolated, substantially pure clinical grade,functional, human monocytes produced in suspension in polypropylenewares in a sterile, serum-free medium and activated with biologicalresponse modifiers and/or adjuvants into AKM and monitoring theirtumoricidal activity in vitro.

An important aspect of the present invention is the use of suchapparatus, containers, appliances, laboratory equipments and the likethat are made of a material which is inert or non-toxic to the humanmonocytes and to which the human monocytes do not adhere or stick. Theuse of non-toxic, non-adherent wares throughout the manipulative steps,in any manner related to the handling of human monocytes, is a criticalfeature of the present invention. A preferred example of an inert,non-toxic, non-adherent and sterilizable material which can be suitablyemployed in accordance with the present invention is polypropylene.Other similar or equivalent materials, other thanpolytetrafluoroethylene, can, of course, be also used so long as suchmaterial is non-toxic and non-adherent to the human monocytes andclinical grade AKM can be produced.

The containers, in accordance with the present invention, are made outof solid polypropylene sheets. Some examples of such containers orlaboratory wares are flasks and micro titer plates and the like ofvarious shapes and sizes, preferably ranging from 0.1 to 200 mlcapacity. For convenience, of course, the shapes and sizes of solidpolypropylene containers are chosen to be similar to other laboratorywares routinely used for testing, culturing or other preparative work.

The term "substantially pure" or "substantially purified" as used hereinmeans that the human monocytes are as pure as it is possible to obtainby standard techniques and methods commonly known to one of the ordinaryskill in the art to which this invention pertains. However, a purity of90 percent or greater is necessary for the monocytes to be substantiallypure.

The term "activated killer monocytes" or AKM as used herein means thatthe isolated monocytes have been exposed to or treated with such agentsor factors which would stimulate, modify or enhance theimmunoregulatory, biological or physiochemical property, nativecharacteristics or functions of the monocytes. Such agents or factorsinclude suitable antigens, adjuvants, biological response modifiers(BRMs) and the like.

The term "inert" container as used herein means that the material ofwhich said container is made has no deleterious or toxic effect on thenatural or normal functions of the monocytes cultured in said container.Polypropylene containers are specifically preferred in this inventionfor obtaining clinical grade AKMs. It is noted that Teflon, which hasbeen suggested by the prior art, is unsuitable for the preparation ofclinical grade AKMs in sufficient amounts (data not shown) in accordancewith the present invention.

The term BRMs include a diverse spectrum of compounds including naturalcytokines such as interferons (IFN), lymphokines such as interleukin-1(IL-1), certain synthetic chemicals with immunomodulatory propertiessuch as polyriboinosinic acid, polyribocytidylic acid, poly L-lysine,carboxymethylcellulose, (poly ICLC) and levamisole; immunomodulatoryadjuvants such as Bacille Calmette Guerin (BCG), Corynebacterium parvum,Staphylococcus protein A, monoclonal antibodies, tumor antigenpreparations and the like. Colony stimulating factors (CSF) andprostaglandin E (PGE) are other examples of suitable BRMs.

Human monocytes can be isolated from the peripheral blood followingroutine techniques well known in the art. However, the preferred methodsand materials employed for isolation and purification of the monocytesare described hereunder. Other preferred methods and materials utilizedare also described. All publications cited hereunder are incorporatedherein by reference.

Isolation of human monocytes

Leukocytes were obtained by leukapheresis (Celltrifuge II leukapheresisapparatus. Travenol Laboratories, Deerfield, Ill.). Monocytes are thenpurified from the unfractionated mononuclear leukocyte preparation bycounter-current centrifugation elutriation (CCE) (Stevenson and Fauci.Manual of Macrophage Methodology; Marcel Dekker, NY. pp. 74-80. 1981).The purity and viability of the monocyte preparations are thendetermined by nonspecific esterase staining, Wright's staining, andlatex bead ingestion as described by Stevenson and Fauci, supra. Theaverage yield of monocytes per normal donor was about 550 million cells(Stevenson, et al. J. Immunol. Meth. 62, 353. 1983).

Culture technique

New culture techniques were developed to allow for maintenance of thesingle-cell suspension state of purified human monocytes, utilizingspecially developed culture plates made of polypropylene. These cultureplates match the exact dimensions of standard 24-well flat-bottomed and96-well round-bottomed polystyrene culture plates (Costar Plastics,Cambridge, Mass.). Human monocytes are counted with an automated cellcounter (Electrozone/Celloscope, Particle Data, Elmhurst, Ill.) andsuspended in serum-free medium or (Roswell Park Memorial Institute,Buffalo, N.Y.) RPMI 1640+10% human serum+L-glutamine at a concentrationof 106 monocytes/ml. For those studies in which human serum wasemployed, no improvement of AKM function was noted when concentrationsin excess of 10% were utilized; therefore, 10% human AB in RPMI 1640serum was used as the standard serum-containing medium. The serum-freemedia employed was as described by Stevenson, et al (T. Immund. Meth.65, 129, 1983) except that beta-mercaptoethanol, which is cytotoxic invivo, was omitted. No antibiotics were added to the cell culturesuspension. Activators of AKM include: Interleukin-2 (IL2) (Cetus),gamma interferon (ISNY) (Genentech) in ranges of 1-1000 units/ml andpolyriboinosinic acid polyribocytidylic acid (poly I:C) (Sigma) inranges of 10-200 mg/ml. Suspension monocyte cultures were placed at 37°C. in a 5% CO₂ incubator either in polypropylene or polystyrene labware.Maximal levels of killing of Indium-111 labelled human tumor targetswere found at 48-72 h of culture, at which time the plates were spunonce at 200×g and the cell culture supernatant was harvested forsubsequent determination of Indium-111 release. All cultures wereperformed in triplicate.

Percent (%) release (AKM tumor cell killing) was calculated according tothe following equation: 100(A/T), where A=cpm released from test welland T-total cpm incorporated into cells. Spontaneous release is %release when target cells alone were incubated. Percent specificAKM-mediated cytotoxicity was obtained by the following equation,100(B-C)/(T-C), where B=cpm released from test well containing monocytesand targets in presence or absence of stimulants (C=cpm released fromtest well containing targets alone in presence or absence of the samestimulant as B). Although C was almost identical to spontaneous release,it was monitored in every experiment; C never exceeded 25%. Lytic unitcalculations were performed as previously described (Stevenson, et al.Am. J. Hematol. 22, 123, 1986).

Statistical methods

For each of the experiments, a linear statistical model which includesall of the main variables (plate type, medium type, and activatingagents) along with main variable interactions was employed. Analysis ofvariance was performed on the raw cytotoxicity data to determinesignificance of main effects. For factors of more than 2 levels,Duncan's multiple range test was employed to determine significance ofpairwise differences (Snedecor and Cochran, Statistical Methods, pp.233-237. 1980).

Effect of culture system variables on AKM tumoricidal activity

The effects of serum-free versus AB serum and polystyrene versuspolypropylene culture plates are summarized in FIG. 1. Serum-free mediumwas shown not to significantly affect the baseline level of tumoricidalactivity in either the adherent (polystyrene) or the suspension(polypropylene) culture systems. In contrast, serum-free mediumsignificantly enhanced AKM tumor cell killing when optimal doses ofactivating agents were used (P=0.03).

It is to be noted that AKM displayed significantly more tumor cellkilling capacity in the polypropylene culture plates (P<0.001) ascompared to polystyrene plates under both AB and serum-free conditions.This is demonstrated by FIG. 2 which shows enhanced attack of tumortargets by AKM in polypropylene (C) as compared to polystyrene (E)plates; actual killing of targets by AKM is also seen in polypropylene(D) versus polystyrene (F) plates. The enhanced killing capacity of AKMmonitored in the described serum-free suspension culture technique wasshown to be applicable to a wide range of human tumors (HT-29, LS 174,SKBR3 and KA(0-3) (FIG. 3). This in vitro technology for generating andmonitoring the tumoricidal function of AKM has allowed the expression ofcytotoxicity data for the first time by the lytic unit method, a datadisplay technique previously only applicable to the killing of tumorcells by lymphocytes. As shown in FIG. 4, this achievement has now beenpossible because of the enhanced reproducibility and dose-response(effector to target ratio) curve data only afforded by the serum-free,suspension culture technology in polypropylene wares.

AKM Therapy of Human Cancer

The object of AKM therapy is to remove substantial numbers of leukocytesfrom the peripheral blood or bone marrow of cancer or immune dysfunctionpatients, followed by the purification of certain cytotoxic leukocytesubsets. Such purified leukocytes are then expanded and/or activated totumoricidal or immunotherapeutic activity in vitro followed byreinfusion of these cells into the sites of tumor burden or immunedysfunction in the patient.

Several clinically relevant criteria must be considered in the design ofany AKM protocol. As shown in Table I, it is important to employ asingle, purified, sterile, toxic-free, cytotoxic leukocyte subset thatis capable of being used in a clinical setting (this includes being notonly sterile but devoid of any toxins). The use of purified leukocytesubsets allows for precise toxicity and physiologic determinations foreach cell type. In the event that the administration of a single celltype is not ameliorative or curative of the immune dysfunction then onecan build upon the single cell type baseline clinical data to designrational "combination activated leukocyte" protocols.

It is important that s sufficient number of cells be obtained to producea clinical effect when infused into the patients (at least 500 million).It is also essential that these cells be maintained in a state ofsuspension to avoid the clinical problems encountered when trying toinfuse clumps of cells. Leukocyte activating substances must be ofclinical grade. Until graft-versus-host disease problems have beenclinically minimized, it is preferred that EVLA protocols are restrictedto the use of autologous leukocytes. A further consideration is that ifin vitro testing demonstrates that the patient's leukocytes haveactivity against his or her tumor cells, mechanisms must be devised toallow for "homing" of the cytotoxic leukocytes to the sites of tumorburden in the patient.

                  TABLE I                                                         ______________________________________                                        Desirable Attributes of AKM Therapy Protocol.                                 ______________________________________                                        A.  A single purified cytotoxic effector cell should be employed.             B.  Should be able to purify effector cells in a manner that                      allows cells to be used clinically (i.e., pyrogen, pathogen                   and toxin-free).                                                          C.  If amplifying and/or activating substances are employed,                      they should be purified and of clinical grade (IND                            # required).                                                              D.  FDA will require a separate approval for the "activated                       effector cell" prior to patient use.                                      E.  Cells should be capable of being cultured in suspension--in                   absence of antibiotics, animal sera or any other sensitizing                  agents.                                                                   F.  Mechanism for obtaining enough effector cells to elicit                       clinical response and/or toxicity should be identified.                   G.  Autologous cells should be employed until graft-vs-host                       disease problem is solved.                                                H.  Mechanism for homing effector cells to tumor site must be                     identified.                                                               I.  In vitro testing should demonstrate activity of purified                      effector cells against patient's tumor cell type.                         ______________________________________                                    

AKM Protocol for Peritoneal Colorectal Carcinomatosis

As mentioned herein supra, a great number of the immunologic effectorcell functions of monocyte/macrophage cell types have been characterizedincluding antigen presentation, the production of certainimmunoregulatory biological response modifiers such as alpha interferon,interleukin 1, colony stimulating factor, and certain criticalcomponents of the complement system. Monocytes and macrophages are alsobelieved to be the predominant cellular component in a number ofcell-mediated immune responses including granuloma formation. Themonocytes and macrophages are also known to be phagocytic and becausethey express Fc receptors for immunoglobulin G, they are majorparticipants in antibody-dependent cellular cytotoxicity reactions(ADCC).

Human AKM also have an ability to recognize and kill tumor targets invitro that is independent of antibody and may be augmented by suchagents as interferon and muramyl dipeptide. Monocytes and macrophagesare major components of the cellular infiltrate of both rodent and humantumors; in vitro studies using tumor-associated macrophages from bothhuman and rodent tumors indicate that these cells can be activated totumoricidal activity with various biological response modifiers. Thereproducible in vitro activity of human blood monocytes, in thesuspension culture system as described herein, is, of course, indicativeof clinical utility.

The present invention is the first to demonstrate clinical feasibilityand efficacy of AKM therapy.

As shown in Table II a number of technical and logistic difficultiesrelevant to the handling of human blood monocytes in the AKM protocolsetting have now been solved. A new technique is described herein forisolating highly purified blood monocytes in large numbers by acombination of two blood component separation techniques: cytapheresis(Stevenson et al, Plasma Ther Transfus. Technol. 4:57-63. 1983, FenwalLaboratories, Deerfield, Ill.), and counter-current centrifugalelutriation (Stevenson, Methods in Enzymology:Immunochemical Techniques,Part G. NY:Academic Press. Beckman Laboratories, Palo Alto, Calif.).Using a combination of these techniques, in accordance with the presentinvention, it has now been possible to obtain greater than 109 humanmonocytes with a purity of 90 percent or more by a negative selectionprocess that allows the cells to remain in suspension. The cells thusobtained are sterile and devoid of antibiotics or any toxins. Utilizingthe methodology described herein supra, sufficient autologous monocytesfrom a single patient are obtained to demonstrate a significant clinicaleffect. Moreover, the ability to "home" these tumoricidal cells to thesite of tumor burden has also been demonstrated in patients withperitoneal colorectal carcinomatosis (Table III-FIG. 5).

Peritoneal-colorectal carcinomatosis (PCC) disease represents ametastatic form of colon cancer and is universally fatal.

                  TABLE II                                                        ______________________________________                                        AKM Therapy                                                                   ______________________________________                                        A.  Techniques for obtaining purified effector monocytes                          identified; counter-current centrifugal elutriation.                      B.  Monocyte purification and activation techniques leave cells                   pyrogen, pathogen and toxin free.                                         C.  Purified, clinical-grade monocyte activating substances                       (gamma IFN) available.                                                    D.  FDA approval for purified gamma interferon activated                          monocytes--granted.                                                       E.  Capable of culturing monocytes in suspension without                          antibiotics, animal sera or other sensitizing agents.                     F.  Mechanism for obtaining enough monocytes for clinical effect                  identified; counter-current centrifugal elutriation.                      G.  Autologous monocytes can be used in an AKM setting.                       H.  Mechanism for "homing" activated monocytes to sites of                        patient tumor burden identified: the peritoneal colorectal                    carcinomatosis.                                                           I.  In vitro testing of monocyte cytotoxicity against various                     human tumor cell types--ongoing.                                          ______________________________________                                    

No effective therapy presently exists. It is believed that the diseasetends to metastasize to distant organs (such as lung and bond) in thelate stage and frequently kills the patient by direct local extensioninto the viscera. This tendency to remain localized suggests thepossibility that local elimination of this metastatic disease maygreatly improve the length and quality of patient life. Previous studieswith these patients in which Tenckhoff catheters have been inserted intothe peritoneal space for the instillation of 5 fluorouracil ("5-FUbellywash" protocol), (Sugarbaker, Principles and Practice of Oncology.Philadelphia: Lippinicott Co., 1982) have provided an excellent clinicalbackground for the direct deposition of tumoricidal leukocytes into thesite of tumor burden in these patients.

The AKM protocol for peritoneal colorectal carcinomatosis, in accordancewith the present invention, is conducted at the National CancerInstitute, Bethesda, Md. As shown in FIG. 5, patients with a diagnosisof peritoneal colorectal carcinomatosis are referred to the NationalCancer Institute for debulking surgery to render the patients asdisease-free as is surgically possible, coupled with the insertion of aTenckhoff catheter which communicates with the peritoneal space.Immediately following surgery, the patient received intraperitonealinfusion of approximately 500 to 900 million AKM. These cells areobtained by a 2-hour cytapheresis procedure followed by purification ofthe monocytes by counter-current centrifugal elutriation.

                                      TABLE III                                   __________________________________________________________________________    AKM Protocol for Peritoneal Colorectal Carcinomatosis.                        __________________________________________________________________________    Week 0    { Debulking surgery/insertion of Tenckhoff catheter                             ↓                                                                      Patient cytapheresis/eluctriation of monocytes                                ↓                                                          Weekly × 16 weeks                                                                   Overnight activation of monocytes in gamma interferon                         ↓                                                                      Infusion of activated monocytes intraperitoneally                             ↓                                                          Week 16   { "Second-look" laparotomy                                                      ↓                                                          Weeks 17-41                                                                             { Maintenance AKM therapy (if indicated)                            __________________________________________________________________________

Following this, the purified monocytes are cultured in suspensionovernight in gamma interferon (Genentech) at a concentration of 1000U/ml. After overnight (about 12-16 hours) activation in gammainterferon, the AKM are infused into the peritoneal space via theTenckhoff catheter. In addition, the patient receives a 2-liter infusionof peritoneal lavage fluid such as Impersol, (Travenol Laboratories,Deerfield, Ill.) to allow for the maximal distribution of the patient'sAKM throughout the peritoneal space. In order to determine withcertainty the patient's response to this form of therapy, a"second-look" laparotomy at the conclusion of 16 weeks of AKM therapy isconducted. Patients who are found to have a complete or partial responseto AKM therapy are then offered a 6-month maintenance AKM therapyregimen.

Clinical Observations Regarding the AKM Protocol

Initially, two PCC patients were treated in the monocyte AKM protocol atthe National Cancer Institute at the Biological Therapeutics and SurgeryBranches. Both patients were remarkably similar in the nature of theirdisease and their response to AKM therapy. Both were white females, 38and 41 years of age; one had a diagnosis of peritoneal colorectalcarcinomatosis for 12 months, the other for 6 months. Both patients werewithout evidence of distant metastatic disease and neither patient hadany other serious illnesses. Their functional status was excellent. Bothpatients had severe involvement of the peritoneal surfaces with cancer;virtually no aspect of the parietal peritoneum was spared. However, bothpatients had visibly less metastatic disease on the small intestine thanelsewhere. Attempts to remove as much grossly visible disease aspossible were successful. One patient required removal of a largesegment of the colon in order to dissect away the malignancy. The otherpatient required removal of the spleen for the same purpose.

Both patients tolerated the AKM therapy remarkably well. As has beenlearned from the second patient, this therapy is safely performed in theoutpatient department following the patient's recovery from the initialdebulking surgery. Typically, the patients arrive one morning in thecytapheresis unit outpatient department and undergo a 2-hourcytapheresis procedure to remove between 5 and 9×10₉ leukocytes.Following this procedure they are sent home, and that afternoon andevening the patients' monocytes are purified by elutriation and placedin suspension culture with gamma interferon (1000 U/ml). The nextmorning, the patients return to the outpatient department to receive theinfusion of their AKM along with 2 liters of additional intraperitonealImpersol; this infusion generally takes approximately 30 minutes. Thepatients then return home with oral analgesics and Tylenol. Generally,within 4 to 6 hours of the infusion of the AKM, the patients note theonset of a low-grade fever (consistently less than 101° F.) and alow-grade abdominal pain. The fever is manageable with Tylenol, and thepain is usually manageable with oral analgesics (occasionally patientshave received parenteral narcotics in the outpatient department if thepain was too severe). Within 12 hours, both the low-grade fever andabdominal pain had generally subsided, and both patients had spent therest of the week performing their normal daily activities. In bothpatients, a low-grade granulocytopenia after the first 4 to 7cytapheresis procedures (total leukocyte count approximately 3,500) isnoted; at this juncture, the frequency of the AKM treatments is adjustedto once every other week with normalization of the peripheral leukocytecount.

Midway through the protocol, the trafficking pattern of theintraperitoneally infused Akm by prelabeling them with ¹¹¹ Indium isanalyzed. FIG. 6 shows the typical distribution of the ¹¹¹Indium-labeled AKM throughout the peritoneal space. The distribution ishomogeneous with the few patchy areas of decreased uptake shown atsecond-look surgery to be due to postoperative adhesions. Wheninterpretable images from these patients up to 5 days afterintraperitoneal infusion of ¹¹¹ In-labeled monocytes is obtained, theevidence indicates that these cells do not traffic outside of theperitoneal space. Instead, they appear to become incorporated in thecellular matrix of the peritoneum, most likely transforming into tissuemacrophages.

Both of the above-cited patients have undergone the "second-look"laparotomy staging procedure. Both were found to have normalization ofthe majority of the surface of their peritoneum including the sites mostheavily infiltrated with tens of thousands of metastatic lesions at thefirst surgery. Neither of the patients were found to have any bulkylesions of the colon or the viscera, nor were they found to have distantmetastases. However, they both had very small amounts of progressivedisease (PD) in places which were restricted (predominantly byperitoneal adhesions) from access to the monocytes. These patients wererendered disease-free by removing the adhesions, exposing these areas,and surgically excising the lesions at operation (O) (all<1 cm.). Theywere thus designated as having an "O-PD" status. Both patients then wenton to receive maintenance therapy following recuperation from thesecond-look laparotomy procedure. Both patients have remained free ofrelapse for over three years. As summarized in Table IV, four additionalPCC cancer patients have been treated with AKM therapy; two continue toenjoy freedom from local relapse for over two years.

The results noted above clearly indicate that immunotherapy ofperitoneal colorectal carcinomatosis with AKM is a feasible andefficacious procedure. In order to obtain maximal antitumor effect theAKM can also be combined with such factors as natural killerlymphocytes, antigen-specific killer T lymphocytes, B-cells and thelike. Such "combination activated leukocyte therapy" can replace,supplement, mimic or reconstruct the natural immunologic system.

Of course, the availability of in vitro cultured, autologous, AKM insuspension opens a new vista for immunotherapy and/or fortification andsupplementation of immune regulatory system in the mammals where suchsystem has been adversely effected due to immune dysfunction.

                                      TABLE IV                                    __________________________________________________________________________    RESULTS OF SEVEN PCC.sup.a PATIENTS TREATED                                   ON THE AKM.sup.b PROTOCOL                                                                                         FREEDOM FROM                              AGE/SEX                                                                             STIMULATOR                                                                             TOXICITIES  RESULTS  LOCAL RELAPSE                             __________________________________________________________________________    44/F  nIFN γ.sup.1                                                                     Tmax = 101  O-PD.sup.2                                                                             3.5 yrs. post-therapy                                    peritoneal irritation                                                         (grade 2).sup.c                                                41/F  nIFN γ                                                                           Afeb.: peritoneal                                                                         O-PD     3.0 yrs. post-therapy                                    irritation (grade 1):1                                                        episode-bacterial                                                             peritonitis                                                    52/M  nIFN γ                                                                           Afeb.: peritoneal                                                                         I-PD.sup.3, 8 cycles                                              irritation (grade 1)                                                          episode-bacterial                                                             peritonitis                                                    42/F  nIFN γ                                                                           Afeb.: peritoneal                                                                         I-PD, 8 cycles                                                    irritation: (grade 1)                                          46/M  nIFN γ                                                                           Tmax = 100: peritoneal                                                                    1 lung metastasis                                                                      2.5 yrs. post-therapy                                    irritation (grade 1)                                                                      post-therapy, no                                                  epidose-bacterial                                                                         clinical peritoneal                                               peritonitis disease                                            31/F  rIFN γ.sup.4                                                                     Tmax = 100: peritoneal                                                                    O-PD     2.0 yrs. post therapy                                    irritation (grade 2)                                           38/M  rIFN γ                                                                           Afeb.: peritoneal                                                                         SD.sup.5, 16 cycles                                               irritation (grade 0)                                           __________________________________________________________________________     .sup.a PCC, peritoneal colorectal carcinomatosis; .sup.1 nIFN γ,        natural interferon γ;                                                   .sup.b AKM, Activated Killer Monocytes; .sup. 2 OPD, small areas of           progressive disease found at 2nd surgery (in areas to which monocytes had     limited access) Pt. rendered surgically diseasefree; .sup.3 IPD,              peritoneal progressive disease (surgically inoperable); .sup.4 rIFN           γ, recombinant interferon γ; .sup.5 SD, stable disease.           .sup.c Peritoneal irritation grading system: 0 = none; 1 = manageable wit     oral analagesics; 2 = requiring parenteranarcotics; 3 = requiring             intravenous fluids and nasogastric suction.                              

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application and thescope of the appended claims.

What is claimed is:
 1. A method of producing activated killer monocytes,comprising the steps of:(a) isolating purified human monocytes byelutriation in a serum free medium in polypropylene containers; and (b)converting the monocytes obtained in step (a) with an effective amountof gamma interferon to produce clinical grade activated killermonocytes.
 2. A method of treating colorectal cancer in patients in needof immunotherapy, comprising peritoneally administering to said patientan immunotherapeutic effective amount of activated killer monocytesproduced by the method of claim 1.